PROJECT SUMMARY The ?nutrient-sensing? enzyme SIRT1 lays at the crossroads of a complex array of molecular interactions that impact susceptibility to diseases as diverse as obesity, diabetes, neurodegeneration, and cancer. One promising therapeutic strategy for the treatment of obesity-related morbidity involves small-molecule STACs, which bind the SIRT1 N-terminal region to allosterically increase SIRT1 activity, thereby elevating the expression of catabolic PGC-1?/PPAR? target genes that help to protect against diet-induced obesity. STACs bind SIRT1 in a 3-helix bundle located at the distal part of the SIRT1 N-terminal region. This 3-helix bundle is shielded by an upstream element of the SIRT1 N-terminal region, protecting the enzyme from an unidentified cellular regulator. Thus, identification of cellular proteins that control enzyme activity through interaction with the 3-helix bundle is key to understanding SIRT1 regulation. Our published and preliminary data suggest PACS-2 is one such SIRT1 regulator and that PACS-2, DBC1 and SIRT1 form a novel tripartite hub that controls SIRT1 deacetylase activity. DBC1 binds to the N-terminal region of SIRT1, where it promotes PACS-2 recruitment and binding to SIRT1. PACS-2 destabilizes the critical 3-helix bundle, which inhibits SIRT1 activity and consequently represses SIRT1-dependent activation of PGC-1?/PPAR? target genes. Our long-term goal is to understand how SIRT1 is regulated to control energy homeostasis in humans. The objective of this particular application is to determine how PACS-2 and DBC1 synergize to allosterically modulate SIRT1 enzyme activity and how this regulatory hub controls the response to fasting or overnutrition in vivo. Our central hypothesis is that disruption of the SIRT1/PACS-2 or SIRT1/DBC1 interactions will prevent high-fat- diet-induced repression of SIRT1 and, therefore, protect mice from hepatic steatosis and insulin resistance. Guided by strong preliminary data, we will test our hypothesis by pursuing three specific aims: 1) Determine the conformational and mechanistic steps by which the PACS-2/SIRT1 interaction regulates enzyme activity; 2) Determine how synergistic actions by PACS-2 and DBC1 inhibit SIRT1; and 3) Determine the physiologic importance of interactions between PACS-2, DBC1, and SIRT1 on hepatic metabolism. The approach is innovative because it will characterize, from the atomic structure to whole-organism function, a previously unrecognized regulatory hub controlling energy metabolism during fasting and overnutrition. This research is significant because it will uncover how SIRT1 regulators control enzyme activity and how we can influence their functions to improve health.